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PST-Gold nanoparticle as an effective anticancer agent with immunomodulatory properties
Colloids and Surfaces B: Biointerfaces 104 (2013) 32–39
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
PST-Gold nanoparticle as an effective anticancer agent with immunomodulatory properties Manu M. Joseph a , S.R. Aravind a , Sheeja Varghese a , S. Mini b , T.T. Sreelekha a,∗ a b
Laboratory of Biopharmaceuticals, Division of Cancer Research, Regional Cancer Centre (RCC), Trivandrum 11, Kerala, India Department of Biochemistry, University of Kerala, Trivandrum, Kerala, India
a r t i c l e
i n f o
Article history: Received 28 August 2012 Received in revised form 5 November 2012 Accepted 29 November 2012 Available online 17 December 2012 Keywords: Polysaccharide Tamarindus indica PST-Gold nanoparticles Immunomodulation Cancer
a b s t r a c t Polysaccharide PST001, which is isolated from the seed kernels of Tamarindus indica (Ti), is an antitumor and immunomodulatory compound. Gold nanoparticles have been used for various applications in cancer. In the present report, a novel strategy for the synthesis and stabilization of gold nanoparticles using anticancer polysaccharide PST001 was employed and the nanoparticles’ antitumor activity was evaluated. PST-Gold nanoparticles were prepared such that PST001 acted both as a reducing agent and as a capping agent. PST-Gold nanoparticles showed high stability, no obvious aggregation for months and a wide range of pH tolerance. PST-Gold nanoparticles not only retained the antitumor effect of PST001 but also showed an enhanced effect even at a low concentration. It was also found that the nanoparticles exerted their antitumor effects through the induction of apoptosis. In vivo assays on BALB/c mice revealed that PST-Gold nanoparticles exhibited immunomodulatory effects. Evaluation of biochemical, hematological and histopathological features of mice revealed that PST-Gold nanoparticles could be administered safely without toxicity. Using the polysaccharide PST001 for the reduction and stabilization of gold nanoparticles does not introduce any environmental toxicity or biological hazards, and these particles are more effective than the parent polysaccharide. Further studies should be employed to exploit these particles as anticancer agents with imaging properties. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Despite advancement in the prevention, diagnosis and treatment of cancer, it is still the leading cause of mortality worldwide. Although chemotherapy regimens represent a major treatment modality, they are accompanied by undesirable side effects due to the severely toxic nature of these chemicals. Therefore, novel pharmaceutical agents with improved specificity and efficacy that are also nontoxic to normal cells would be of immense clinical value. Polysaccharides are a group of widely occurring natural biological macromolecules with tremendous structural diversity, and their biological activities have attracted much attention in medicine [1,2]. Antitumor, immunomodulatory, antimicrobial, antiulcer, antioxidant and several other pharmacological activities from various polysaccharides have been reported [3–5]. Tamarindus indica (Ti), a tree from the family of Leguminosae, is widely grown in India and other Asian countries, and its components are utilized in daily human life. Polysaccharide isolated from the seed kernels of Ti mainly consists of xyloglucans, which have been shown to possess various pharmacological properties.
∗ Corresponding author. Tel.: +91 4712522378; fax: +91 4712447454. E-mail address: [email protected] (T.T. Sreelekha). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.11.046
They have been used as anticancer, immunomodulatory, antiviral, antioxidant and even artificial tear agents in modern medicine [6–11]. Ti polysaccharide possess various properties, including biocompatibility, biodegradability, high viscosity, high thermal stability, broad range of pH tolerance and adhesiveness, that facilitate their broad usages as stabilizers, thickeners, gelling agents, binders and additives in food and pharmaceutical industries; they are also used for controlling drug release in pharmaceutical applications [9]. Gold nanoparticles (AuNPs) have recently emerged as an attractive candidate for targeted delivery of various therapeutic agents [12,13] due to their distinctive features, including reasonably low cytotoxicity, tunable surface characteristics and stability under in vivo conditions. AuNPs selectively accumulate in tumor cells, showing bright-light scattering, and hence can serve as specialized microscopic probes to study cancer cells [14]. The conventional methods for the synthesis of AuNPs involve toxic chemicals [15], but the use of natural materials, including plants, algae and microbes, have also been reported [16–19]. Recently, certain polysaccharides, such as sucrose, cellulose and chitosan, have also been used for the synthesis and stabilization of AuNPs [20,21]. These approaches would not introduce environmental toxicity or biological hazards. The polysaccharide PST001, which is extracted from the seed kernels of T. indica, was previously isolated and characterized by our
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laboratory; it was found to be an anticancer agent with excellent in vitro and in vivo immunomodulatory properties and no toxicity to normal cells [6–8]. The aim of the present study was to combine effective delivery of the antitumor and immunomodulatory activities of PST001 by generating AuNPs using the polysaccharide. 2. Materials and methods 2.1. Isolation and purification of polysaccharide PST001 from seed kernels of Tamarindus indica Ripened seeds of Ti were obtained from the Thiruvananthapuram District of Kerala. The seeds were dried and powdered, and then the polysaccharide PST001 was isolated as previously reported [6,22]. The isolated polysaccharide was purified with gel filtration chromatography using Sephadex G-200; 0.001 M phosphate buffered saline (PBS) was used as the eluent, and the purified product was lyophilized. The total carbohydrate content was determined by Dubois’s method [23] using d-glucose as the standard. 2.2. Preparation and characterization of PST-Gold nanoparticles After the isolation of polysaccharide, the nanoparticles were prepared. The preparation of AuNPs using PST001 was performed based on earlier reports [20,21,24,25], with modifications. All glassware used was cleaned with freshly prepared aqua regia solution (HCl:HNO3 3:1) and rinsed thoroughly with distilled water prior to use. To 1 mM solution of HAuCl4 (1 ml), 3 ml of 10 mg/ml solution of PST001 was added drop-wise with constant stirring on a magnetic stirrer plus hotplate heated to 70 ◦ C; the process continued for 2–3 h until an intense red-colored solution was obtained. The particles thus formed were named PST-Gold nanoparticles. Capping agents can be used to stabilize the nanoparticles and prevent aggregation, but no external capping agents were added in this case. The PST-Gold nanoparticles were initially characterized with UV–vis spectroscopy (Bio Spec-1601, Shimadzu), transmission electron microscopy at an accelerated voltage of 80 kV (Hitachi TEM system) and finally with dynamic light scattering (Malvern DLS instrument V2.0). 2.3. Stability assay of PST-Gold nanoparticles Once the nanoparticles were prepared, their stability was evaluated by spectroscopy over the parameters of time and pH at ambient temperature. In the pH stability study, the pH of PST-Gold nanoparticles was adjusted using 0.1 N hydrochloric acid (pHs 1, 2, 3, 5 and 6) and 0.1 M sodium hydroxide (pHs 12 and 14) on a calibrated pH meter (Cyber Scan 510). 2.4. Cell cultures The human cancer cell lines MCF-7 (breast cancer), K562 (leukemia) and A549 (adenocarcinoma) were obtained from the National Centre for Cell Science, Pune, India. A375 (melanoma), HepG2 (hepatocellular carcinoma) and HCT116 (colon cancer) cells were kindly provided by RGCB (Rajiv Gandhi Centre for Biotechnology), Thiruvananthapuram, India. The cells were maintained in DMEM media with 10% fetal bovine serum at 37 ◦ C and 5% CO2 in an incubator (Heraeus BB 15). 2.5. Cytotoxicity assay The growth inhibition capacity of PST-Gold nanoparticles was evaluated on cancer cell lines and isolated normal lymphocytes by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) assay as previously reported [3,6]. This assay’s function is based on
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the cleavage of tetrazolium salt by mitochondrial dehydrogenases in viable cells. The absorbance was measured at 570 nm using a microplate spectrophotometer (BioTek Power Wave XS). The proliferation rate and inhibitory rate of the cells were calculated with the following formulas: Proliferation rate (PR) % = [Abs sample/Abs control] × 100; Inhibitory rare (IR) % = 100 − PR. MTT assays were performed on cancer cell lines and lymphocytes containing PST-Gold nanoparticles, PST001 and standard citrate-capped AuNPs (Sigma G1652) that are of the same size as PST-Gold nanoparticles.
2.6. Acridine orange–ethidium bromide staining assay Acridine orange–ethidium bromide dual staining is the most commonly used method to detect apoptosis based on the differential uptake of two fluorescent DNA binding dyes by viable and nonviable cells [26]. The experiment was performed as described previously [7]. The cells were observed under an inverted fluorescent microscope under an FITC filter (Olympus 1X51).
2.7. Flow cytometric evaluation of Annexin V–FITC staining Annexin V–FITC staining assay was performed with FITC Annexin V Apoptosis Detection Kit (BD Pharmingen #556547, BD Biosciences, San Jose, CA) as per the manufacturer’s instructions and as previously described [7]. FITC-conjugated Annexin V, which binds to phosphatidylserine, was detected using a FACS Calibur flow cytometer (BD) and analyzed with the CellQuest Pro software.
2.8. In vivo toxicity studies Evaluation of compound toxicity in a biological system requires special attention. The effect of PST-Gold nanoparticles on in vivo systems was evaluated by acute and subacute toxicity assays on 5- to 6-week-old male BALB/c mice as previously described [6]. Briefly, for acute toxicity studies, the compound was administered intraperitoneally (ip) up to a concentration of 2000 mg/kg body weight. For subacute toxicity assays doses of the nanoparticles corresponding to various fractions of the LD50 (1/5, 1/10 and 1/20) were prepared and administered intraperitoneally to each group of mice for 14 consecutive days, whereas the control group was treated with the vehicle (PBS) only. On the 15th day, the animals were sacrificed by cervical dislocation; blood, femur bones and internal organs were collected and used for further evaluation [27]. All animal studies were performed in accordance with the Institutional Animal Ethical Committee’s (IAEC) approval.
2.9. In vivo immunomodulation studies An immunomodulator is a substance that affects the immune system. Lymphocytes were isolated from the blood 14 days after administration of the compound. Its proliferation status was evaluated with the MTT assay as described before, and the status of CD3+, CD4+ and CD8+ cells was determined by flow cytometry and CellQuest Pro [6]. Various biochemical and hematological parameters were also evaluated. Femur bones were collected and cut at the level of epiphyseal plates of the proximal and distal ends of the bone. Bone marrow cells were aspirated after flushing with RPMI media, and cell counts were calculated as described previously [6].
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2.10. Statistical analysis Data were expressed as the mean ± standard deviation (SD) of three replicates and were analyzed using GraphPad PRISM software version 5.0 (GraphPad Software, USA). One-way analysis of variance was used for repeated measurements, and the differences were considered to be statistically significant if P < 0.05. The IC50 values were calculated using the Easy Plot software. 3. Results and discussion 3.1. PST-Gold nanoparticles were prepared and are stable The polysaccharide PST001 isolated from the seed kernels of Ti was found to be pH neutral, and the total sugar content was 98%, as determined by the phenol–sulfuric acid method. PST0001 was purified by gel filtration chromatography, lyophilized and stored. We found that PST001 successfully reduced HAuCl4 to form gold nanoparticles with a deep red color (Fig. 1a). PST001 not only acted as a reducing agent for the production of gold nanoparticles but also served as a capping agent, imparting stability to the PST-Gold nanoparticles and preventing their re-aggregation without the use of any other capping agent. The successful production of PST-Gold nanoparticles was evaluated using UV–vis spectroscopy, which produced the characteristic peak of gold nanoparticles approximately 550 nm (Fig. 1b); PST001 and HAuCl4 failed to produce any characteristic peak from 350 to 750 nm. At an accelerated voltage of 80 kV, TEM evaluation of the nanoparticle size clearly showed that the majority of the particles were circular in shape with an average size of 15–20 nm (Fig. 1c). This result was confirmed using DLS, which also indicated that the particles have an average size of 20 nm (Fig. 1d). Although PST-Gold nanoparticles do not produce any visible aggregation, their stability was evaluated for various time intervals (Fig. 1e) and they remained stable even up to 12 months. Evaluation of nanoparticle stability over a wide range of pH using UV–vis spectroscopy clearly demonstrated a high tolerance for pH change (Fig. 1f) with no significant shift in the position of the peak over a pH range of 1–14. The present study investigated the preparation of gold nanoparticles using the water soluble, anticancer polysaccharide PST001 isolated from the seed kernels of T. indica and further evaluated their biological potential as a cytotoxic agent specific to cancer cells. Water soluble polysaccharides were found to be therapeutically applicable for various pharmacological applications, including the use as drug carriers [28]. Gold nanoparticles have a long history of use in various pharmacological applications [29–31]. The production of PST-Gold nanoparticles using PST001 does not include the use of any hazardous chemicals and environmental toxins and hence is a “green” nanoparticle synthesis process [32]. This process also does not require the use of a capping agent because PST001 itself acts as a capping agent in addition to its role as a reducing agent. PST-Gold nanoparticles have an average size of 20 nm and are stable for up to 1 year with a wide range of pH tolerance. Therefore, they are more stable than commercially available borohydrate- or citrate-reduced gold nanoparticles [15]. 3.2. Anticancer effect of PST-Gold nanoparticles Because PST001 is reported to be an anticancer agent with immunomodulatory properties, the PST-Gold nanoparticles were also evaluated for their anticancer potential on various cancer cell lines. This potential was found to be highly significant (P < 0.001) in all cell lines examined. Cytotoxicity of PST-Gold nanoparticles was also evaluated in the cancer cell lines. Breast cancer cell line MCF7 and leukemia cell line K562 were growth-arrested with
IC50 values of 70.3 ± 1.2 g/ml and 48.9 ± 1.8 g/ml, respectively, after 48 h of incubation with the nanoparticles, whereas the native polysaccharide failed to inhibit 50% of cell growth even at a higher concentration and with a longer incubation period (Fig. 2a and b, and Supplementary Fig. 1a and b) in both cell lines. The cytotoxic potential of the nanoparticles increased in a dose-dependent manner up to 100 g/ml in both cell lines. PST-Gold nanoparticles also exhibited excellent cytotoxic potential against A549 (adenocarcinoma), A375 (melanoma), HepG2 (hepatocellular carcinoma) and HCT116 (colon cancer) cells. IC50 values of the nanoparticles were 53.4 ± 0.9 g/ml and 33.8 ± 1.1 g/ml at 48 h for A549 and A375 cells, respectively, whereas the IC50s for PST001 in the two cell lines were 82.03 ± 1.6 g/ml and 61 ± 1.7 g/ml, respectively, after 72 h of incubation. PST-Gold nanoparticles exhibited a dose-dependent increase in cytotoxicity in A375 cells but reached optimum cytotoxicity at 100 g/ml in A549 cells (Fig. 2c and d, and Supplementary Fig. 1c and d). The cytotoxic potential increased in a dose-dependent fashion up to 10 g/ml for HepG2 cells, and an IC50 value of 6.5 ± 0.5 g/ml was obtained after 48 h of incubation. In HepG2 cells, the IC50 dose of PST001 was 33.7 ± 1.3 g/ml after 72 h of incubation (Fig. 2e, and Supplementary Fig. 1e). In HCT116 cells, PST-Gold nanoparticles also exhibited a dose-dependent increase in cytotoxicity, with an IC50 of 50.07 ± 1.5 g/ml at 48 h, whereas the IC50 of PST001 was 82 ± 1.2 g/ml at 72 h (Fig. 2f, and Supplementary Fig. 1f). Standard citrate-capped gold nanoparticles of 20 nm in size and 1 mM HAuCl4 were found to be nontoxic in all of these cell lines. To evaluate the cytotoxic potential of PST001 in normal cells, an MTT assay was performed on isolated normal lymphocytes. PST-Gold nanoparticles were found not only to be nontoxic to the cells across all concentrations but also strongly immunostimulatory. A proliferative index (PI) of 1.19 was obtained at a concentration of 0.1 g/ml. Standard gold nanoparticles showed little toxicity at higher concentrations but exhibited no immunostimulatory effects (Fig. 2g). Earlier we showed that PST001 alone was much less cytotoxic to cancer cells than PSTGold nanoparticles. Because PST001 was found to be nontoxic to normal cells, the nanoparticles were also checked for cytotoxicity on isolated normal lymphocytes. We found that, in addition to being nontoxic, PST-Gold nanoparticles also exerted immunostimulatory effects with a PI of up to 1.19 on the lymphocytes. Various polysaccharides were previously reported to have both anticancer and immunomodulatory effects [1,3,6]; however, the present study revealed that PST-Gold nanoparticles are more potential than other compounds. 3.3. PST-Gold nanoparticles exert anticancer effects through induction of apoptosis Because PST-Gold nanoparticles exhibited significant cytotoxicity specifically against cancer cells, their mode of cell death induction was evaluated using various apoptotic assays. Membrane blebbing, which is a hallmark of apoptosis, refers to irregular bulges in the plasma membrane of a cell caused by localized decoupling of the cytoskeleton from the plasma membrane. Morphological evaluation of the cell lines treated with PST-Gold nanoparticles at a concentration of 10 g/ml for 48 h using phasecontrast microscopy revealed a decrease in the number of cells exhibiting morphological features of apoptosis, such as distorted shape and membrane blebbing in comparison with the control group (Supplementary Fig. 2). Using acridine orange–ethidium bromide staining, cells treated with PST-Gold nanoparticles showed a change in color from green to yellow/red with associated apoptotic features such as membrane blebbing, nuclear condensation and presence of apoptotic bodies compared to the control (Fig. 3, insert). PST-Gold-induced apoptosis was confirmed by flow cytometry analysis of Annexin V staining. There was a significant
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Fig. 1. Preparation and stability assay of PST-Gold nanoparticles. (a) Photographic image of freshly prepared PST-Gold nanoparticles. (b) UV–vis spectra of PST-Gold nanoparticles, HAuCl4 and PST001. (c) TEM images of PST-Gold nanoparticles, magnified 20,000×. (d) DLS spectra of PST-Gold nanoparticles. (e) Stability assay of PST-Gold nanoparticles in various time intervals and (f) in various pH conditions. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
(P < 0.001) increase in the Annexin V positive cells in all of the cell lines treated with 10 g/ml PST-Gold nanoparticles for 48 h (Supplementary Table 1). In MCF 7 cells, the controls exhibited 2 ± 0.07% Annexin V positive cells, whereas compound treated cells were 41.4 ± 1.5% positive (Fig. 3a and b). A similar pattern was observed for K562, A549 and A375 cell lines, where the control cells were 4.5 ± 0.9%, 11 ± 1.2% and 16 ± 1.1% Annexin V positive and PST-Gold nanoparticle-treated cells were 8.3 ± 0.9%, 35 ± 1.6% and 51 ± 1.9% positive, respectively (Fig. 3c–h). The percentages of apoptotic HepG2 and HCT116 cells significantly increased from 3.5 ± 0.6% and 9.9 ± 1.3% in the controls to 47 ± 2.1% and 32.5 ± 2.2% in nanoparticle-treated cells, respectively (Fig. 3i–l). The enhanced activity exhibited by the nanoparticles compared with the parent polysaccharide might be due to the increased uptake of the particles via endocytosis because of their smaller size and increased surface to volume ratio [33]. 3.4. In vivo evaluation of PST-Gold nanoparticles Even though PST-Gold nanoparticles exhibited significant cytotoxicity against cancer cells and immunomodulatory effects in vitro, their activity in biological systems requires further evaluation. In vivo toxicity of PST-Gold nanoparticles was assessed on 5- to 6-week-old male BALB/c mice. There were no behavioral changes or visible toxicity symptoms upon ip administration of PST-Gold up to a concentration of 2000 mg/kg; hence, the LD50 was taken to be 2000 mg/kg. After the administration of the compound at different concentrations for 14 consecutive days, animals were sacrificed by cervical dislocation and various parameters were analyzed. None of the PST-Gold nanoparticle-treated animals from any
of the groups exhibited weight loss throughout the duration of the experiment. Various biochemical and hematological parameters of the sacrificed animals were assessed for any toxicity induced by the nanoparticles (Supplementary Table 2). There was an increase in the RBC and WBC count in the groups administered with the compound, regardless of the concentration, which again reinforced the immunostimulatory effects of PST-Gold nanoparticles. There was no significant variation in any of the common biochemical parameters for the compound-treated mice compared with the control; moreover, no toxicity was observed to be associated with the administration of the compound. Hematoxylin–eosin staining was performed on the livers, kidneys, lungs, hearts and spleens of the sacrificed animals to evaluate any pathological changes associated with compound administration at the organ level. No significant pathological changes were observed with the heart, lung and spleen in any of the PST-Gold-treated mice compared with the control group (Supplementary Fig. 3). PST-Gold nanoparticles administered at a concentration of 100 mg/kg appeared to have no effect on the liver and kidney (Fig. 4c and d), and mice treated with 200 and 400 mg/kg compound exhibited slight cytological abnormalities. The hepatocytes appeared to be slightly swollen in morphology with enlarged cytoplasm and normal nuclei in the mice given 200 mg/kg compound (Fig. 4f); the shape of the cells appeared to be irregular at the higher concentration (Fig. 4h), but the nuclei remained normal. Although the glomeruli appeared normal for the mice given 200 mg/kg PST-Gold nanoparticles, the cells lining the renal tubules showed slight changes with a swollen morphology (Fig. 4e). Similar to the changes in the glomeruli, the tubular cells also appeared larger with a smaller lumen (Fig. 4g).
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Fig. 2. MTT assay on cancer cell lines and lymphocytes. (a) Cytotoxicity profile on 48-h incubation of PST-Gold nanoparticles, PST001 and standard citrate capped gold nanoparticles on MCF-7 cells, (b) on K562 cells, (c) on A549 cells, (d) on A375 cells, (e) on HepG2 cells, (f) on HCT116 cells and (g) on isolated normal lymphocytes at 72 h of incubation. Results are expressed as the mean ± SD.
Compound-treated mice exhibited a significant (P < 0.001) increase in the lymphocyte proliferation status both at the time of sacrifice (0 h) and at 72 h after in vitro incubation in comparison with the control group (Fig. 5a). Although all concentrations of the nanoparticles showed significant immunomodulatory activity, the mice given with 100 mg/kg PST-Gold nanoparticles showed maximum lymphocyte proliferation with PIs of 1.8 ± 0.01 and 1.6 ± 0.08 at 0 h and 72 h, respectively. Immunophenotyping of lymphocyte subsets with CD markers (CD3, CD4 and CD8) showed an increase in the proportion of the T lymphocyte population in the PSTGold nanoparticle-treated mice compared with the control group
(Fig. 5b). Mice given 100 mg/kg compound exhibited a significant increase in all of the T lymphocyte subsets, with PIs of 48.7 ± 4.5%, 35.7 ± 4% and 11.2 ± 2% for CD3, CD4 and CD8 subtypes, respectively. In comparison, the PIs in the control group were 22.8 ± 3.1%, 24.5 ± 3% and 3.3 ± 0.3% for CD3, CD4 and CD8 subtypes, respectively. The elevated T cell population serves as an index for the immunostimulatory property of PST-Gold nanoparticles. The bone marrow cellularity was determined by counting the number of bone marrow cells from the femur bones of the sacrificed animals. The group administered with 100 mg/kg PST-Gold showed the maximum bone marrow cell count at 8 × 106 cells/femur, whereas
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Fig. 3. Apoptotic evaluation of cell lines treated with 10 g/ml PST-Gold nanoparticles for 48 h by Annexin V–FITC staining assay. The insert figure shows acridine orange–ethidium bromide staining images of the same on corresponding cell lines. MCF 7 cells (a) control and (b) PST-Gold. K562 cells (c) control and (d) PST-Gold. A549 cells (e) control and (f) PST-Gold. A375 cells (g) control and (h) PST-Gold. HepG2 cells (i) control and (j) PST-Gold. HCT116 cells (k) control and (l) PST-Gold. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
the control group produced 4.8 × 106 cells/femur. Notably, the bone marrow cell counts were significantly higher (P < 0.001) in the PST-Gold treated mice than in the control group across all concentrations (Fig. 5c). Acute toxicity assay performed on nanoparticle-treated BALB/c mice did not show any lethal effect, and the LD50 was taken
to be 2000 mg/kg. Histopathological evaluation of various organs after hematoxylin–eosin staining demonstrated no pathological symptoms for the heart, lung and spleen, but slight changes were observed with the liver and kidney in mice administered with 200 and 400 mg/kg nanoparticles. However, analysis of various biochemical parameters revealed no abnormality compared with
Fig. 4. Light microscopic images of H&E staining of various organs of BALB/c mice after 14 days administration of PST-Gold nanoparticles and normal saline. Control (a) kidney and (b) liver. 100 mg/kg PST-Gold (c) kidney and (d) liver. 200 mg/kg PST-Gold (e) kidney and (f) liver. 400 mg/kg PST-Gold (g) kidney and (h) liver.
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Fig. 5. Immunomodulatory effects of PST-Gold nanoparticles on BALB/c mice after 14 days of administration of the compound. (a) Lymphocyte proliferation status of the sacrificed animal at the hour of sacrifice (0 h) and after 72 h. (b) Immunophenotyping for the status of lymphocyte subsets. (c) Bone marrow cellularity from the bone marrow cells of femur bones. Results are expressed as the mean ± SD. Statistically significant differences are at **P < 0.01, ***P < 0.001, ns is the non-significant, as compared with the control group.
the control mice. The slight pathological abnormalities observed with the liver and kidney at higher concentrations of nanoparticles were expected to be reversible. The in vivo immunomodulatory effects of PST-Gold nanoparticles were further demonstrated by significant increases in the lymphocyte proliferation status, bone marrow cell count and T lymphocyte population in mice treated with the nanoparticle for 14 days relative to the controls. Conventional chemotherapeutic agents often suppress the immune system of the host [34], which warrants the search for anticancer agents that are nontoxic to normal cells. Gold nanoparticles have been evaluated in diverse applications, including in vitro assays, in vitro and in vivo imaging, cancer therapy and drug delivery. Tumor-targeting technologies that exploit gold’s inherent biocompatibility are being developed to deliver drugs directly into cancerous tumors. Additionally, simple, cost-effective and sensitive diagnostic tests are being developed for the early detection of prostate and other cancers. Furthermore, application of gold nanoparticles in radiotherapy has been reported recently. Roa et al. reported that gold nanoparticle sensitizes prostate cancer cells to radiation by regulating the cell cycle [35]. Gold nanoparticle could also provide advantages in terms of radiation dose enhancement [36], as reported by Wan et al. Therefore, PST-Gold nanoparticle needs to be evaluated for its various applications in cancer treatment and management. 4. Conclusions The preparation of PST-Gold nanoparticles using the anticancer polysaccharide PST001 was found to be of immense therapeutic value because the particles exhibited both immunomodulatory and cytotoxic properties. PST-Gold nanoparticles exerted its cytotoxicity in tumor cells through the induction of apoptosis and were found to be nontoxic to normal tissues in BALB/c mice. Although slight
pathological abnormalities were observed based on H&E staining at higher concentrations of the nanoparticle, the groups given lower doses of the nanoparticle were found to be unaffected. Immunostimulatory effects of PST-Gold nanoparticles on BALB/c mice were more pronounced 14 days after administration of the compound, as demonstrated by a significant increase in bone marrow cell count and CD3/4/8 counts. The data presented here suggest that PST-Gold nanoparticle may be used as an effective anticancer agent with strong immunomodulatory potential. Acknowledgements We greatly acknowledge the Council of Scientific and Industrial Research (CSIR), Govt. of India, for the research fellowship; Kerala State Council for Science, Technology and Environment (KSCSTE), Govt. of Kerala, for the financial support; and the National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, for the DLS analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2012.11.046. References [1] V.E. Ooi, F. Liu, Curr. Med. Chem. 7 (2000) 715. [2] V.R. Sinha, R. Kumaria, Pharm. Int. J. 224 (2001) 19. [3] M.J. Manu, S.R. Aravind, V. Sheeja, S. Mini, T.T. Sreelekha, Mol. Med. Rep. 5 (2012) 489. [4] F. Liu, V. Ooi, S.T. Chang, Life Sci. 60 (1997) 763. [5] G. Franz, Planta Med. 55 (1989) 493. [6] S.R. Aravind, M.J. Manu, V. Sheeja, B. Prabha, T.T. Sreelekha, Sci. World J. (2012) 361382.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
PST-Gold nanoparticle as an effective anticancer agent with immunomodulatory properties Manu M. Joseph a , S.R. Aravind a , Sheeja Varghese a , S. Mini b , T.T. Sreelekha a,∗ a b
Laboratory of Biopharmaceuticals, Division of Cancer Research, Regional Cancer Centre (RCC), Trivandrum 11, Kerala, India Department of Biochemistry, University of Kerala, Trivandrum, Kerala, India
a r t i c l e
i n f o
Article history: Received 28 August 2012 Received in revised form 5 November 2012 Accepted 29 November 2012 Available online 17 December 2012 Keywords: Polysaccharide Tamarindus indica PST-Gold nanoparticles Immunomodulation Cancer
a b s t r a c t Polysaccharide PST001, which is isolated from the seed kernels of Tamarindus indica (Ti), is an antitumor and immunomodulatory compound. Gold nanoparticles have been used for various applications in cancer. In the present report, a novel strategy for the synthesis and stabilization of gold nanoparticles using anticancer polysaccharide PST001 was employed and the nanoparticles’ antitumor activity was evaluated. PST-Gold nanoparticles were prepared such that PST001 acted both as a reducing agent and as a capping agent. PST-Gold nanoparticles showed high stability, no obvious aggregation for months and a wide range of pH tolerance. PST-Gold nanoparticles not only retained the antitumor effect of PST001 but also showed an enhanced effect even at a low concentration. It was also found that the nanoparticles exerted their antitumor effects through the induction of apoptosis. In vivo assays on BALB/c mice revealed that PST-Gold nanoparticles exhibited immunomodulatory effects. Evaluation of biochemical, hematological and histopathological features of mice revealed that PST-Gold nanoparticles could be administered safely without toxicity. Using the polysaccharide PST001 for the reduction and stabilization of gold nanoparticles does not introduce any environmental toxicity or biological hazards, and these particles are more effective than the parent polysaccharide. Further studies should be employed to exploit these particles as anticancer agents with imaging properties. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Despite advancement in the prevention, diagnosis and treatment of cancer, it is still the leading cause of mortality worldwide. Although chemotherapy regimens represent a major treatment modality, they are accompanied by undesirable side effects due to the severely toxic nature of these chemicals. Therefore, novel pharmaceutical agents with improved specificity and efficacy that are also nontoxic to normal cells would be of immense clinical value. Polysaccharides are a group of widely occurring natural biological macromolecules with tremendous structural diversity, and their biological activities have attracted much attention in medicine [1,2]. Antitumor, immunomodulatory, antimicrobial, antiulcer, antioxidant and several other pharmacological activities from various polysaccharides have been reported [3–5]. Tamarindus indica (Ti), a tree from the family of Leguminosae, is widely grown in India and other Asian countries, and its components are utilized in daily human life. Polysaccharide isolated from the seed kernels of Ti mainly consists of xyloglucans, which have been shown to possess various pharmacological properties.
∗ Corresponding author. Tel.: +91 4712522378; fax: +91 4712447454. E-mail address: [email protected] (T.T. Sreelekha). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.11.046
They have been used as anticancer, immunomodulatory, antiviral, antioxidant and even artificial tear agents in modern medicine [6–11]. Ti polysaccharide possess various properties, including biocompatibility, biodegradability, high viscosity, high thermal stability, broad range of pH tolerance and adhesiveness, that facilitate their broad usages as stabilizers, thickeners, gelling agents, binders and additives in food and pharmaceutical industries; they are also used for controlling drug release in pharmaceutical applications [9]. Gold nanoparticles (AuNPs) have recently emerged as an attractive candidate for targeted delivery of various therapeutic agents [12,13] due to their distinctive features, including reasonably low cytotoxicity, tunable surface characteristics and stability under in vivo conditions. AuNPs selectively accumulate in tumor cells, showing bright-light scattering, and hence can serve as specialized microscopic probes to study cancer cells [14]. The conventional methods for the synthesis of AuNPs involve toxic chemicals [15], but the use of natural materials, including plants, algae and microbes, have also been reported [16–19]. Recently, certain polysaccharides, such as sucrose, cellulose and chitosan, have also been used for the synthesis and stabilization of AuNPs [20,21]. These approaches would not introduce environmental toxicity or biological hazards. The polysaccharide PST001, which is extracted from the seed kernels of T. indica, was previously isolated and characterized by our
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laboratory; it was found to be an anticancer agent with excellent in vitro and in vivo immunomodulatory properties and no toxicity to normal cells [6–8]. The aim of the present study was to combine effective delivery of the antitumor and immunomodulatory activities of PST001 by generating AuNPs using the polysaccharide. 2. Materials and methods 2.1. Isolation and purification of polysaccharide PST001 from seed kernels of Tamarindus indica Ripened seeds of Ti were obtained from the Thiruvananthapuram District of Kerala. The seeds were dried and powdered, and then the polysaccharide PST001 was isolated as previously reported [6,22]. The isolated polysaccharide was purified with gel filtration chromatography using Sephadex G-200; 0.001 M phosphate buffered saline (PBS) was used as the eluent, and the purified product was lyophilized. The total carbohydrate content was determined by Dubois’s method [23] using d-glucose as the standard. 2.2. Preparation and characterization of PST-Gold nanoparticles After the isolation of polysaccharide, the nanoparticles were prepared. The preparation of AuNPs using PST001 was performed based on earlier reports [20,21,24,25], with modifications. All glassware used was cleaned with freshly prepared aqua regia solution (HCl:HNO3 3:1) and rinsed thoroughly with distilled water prior to use. To 1 mM solution of HAuCl4 (1 ml), 3 ml of 10 mg/ml solution of PST001 was added drop-wise with constant stirring on a magnetic stirrer plus hotplate heated to 70 ◦ C; the process continued for 2–3 h until an intense red-colored solution was obtained. The particles thus formed were named PST-Gold nanoparticles. Capping agents can be used to stabilize the nanoparticles and prevent aggregation, but no external capping agents were added in this case. The PST-Gold nanoparticles were initially characterized with UV–vis spectroscopy (Bio Spec-1601, Shimadzu), transmission electron microscopy at an accelerated voltage of 80 kV (Hitachi TEM system) and finally with dynamic light scattering (Malvern DLS instrument V2.0). 2.3. Stability assay of PST-Gold nanoparticles Once the nanoparticles were prepared, their stability was evaluated by spectroscopy over the parameters of time and pH at ambient temperature. In the pH stability study, the pH of PST-Gold nanoparticles was adjusted using 0.1 N hydrochloric acid (pHs 1, 2, 3, 5 and 6) and 0.1 M sodium hydroxide (pHs 12 and 14) on a calibrated pH meter (Cyber Scan 510). 2.4. Cell cultures The human cancer cell lines MCF-7 (breast cancer), K562 (leukemia) and A549 (adenocarcinoma) were obtained from the National Centre for Cell Science, Pune, India. A375 (melanoma), HepG2 (hepatocellular carcinoma) and HCT116 (colon cancer) cells were kindly provided by RGCB (Rajiv Gandhi Centre for Biotechnology), Thiruvananthapuram, India. The cells were maintained in DMEM media with 10% fetal bovine serum at 37 ◦ C and 5% CO2 in an incubator (Heraeus BB 15). 2.5. Cytotoxicity assay The growth inhibition capacity of PST-Gold nanoparticles was evaluated on cancer cell lines and isolated normal lymphocytes by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) assay as previously reported [3,6]. This assay’s function is based on
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the cleavage of tetrazolium salt by mitochondrial dehydrogenases in viable cells. The absorbance was measured at 570 nm using a microplate spectrophotometer (BioTek Power Wave XS). The proliferation rate and inhibitory rate of the cells were calculated with the following formulas: Proliferation rate (PR) % = [Abs sample/Abs control] × 100; Inhibitory rare (IR) % = 100 − PR. MTT assays were performed on cancer cell lines and lymphocytes containing PST-Gold nanoparticles, PST001 and standard citrate-capped AuNPs (Sigma G1652) that are of the same size as PST-Gold nanoparticles.
2.6. Acridine orange–ethidium bromide staining assay Acridine orange–ethidium bromide dual staining is the most commonly used method to detect apoptosis based on the differential uptake of two fluorescent DNA binding dyes by viable and nonviable cells [26]. The experiment was performed as described previously [7]. The cells were observed under an inverted fluorescent microscope under an FITC filter (Olympus 1X51).
2.7. Flow cytometric evaluation of Annexin V–FITC staining Annexin V–FITC staining assay was performed with FITC Annexin V Apoptosis Detection Kit (BD Pharmingen #556547, BD Biosciences, San Jose, CA) as per the manufacturer’s instructions and as previously described [7]. FITC-conjugated Annexin V, which binds to phosphatidylserine, was detected using a FACS Calibur flow cytometer (BD) and analyzed with the CellQuest Pro software.
2.8. In vivo toxicity studies Evaluation of compound toxicity in a biological system requires special attention. The effect of PST-Gold nanoparticles on in vivo systems was evaluated by acute and subacute toxicity assays on 5- to 6-week-old male BALB/c mice as previously described [6]. Briefly, for acute toxicity studies, the compound was administered intraperitoneally (ip) up to a concentration of 2000 mg/kg body weight. For subacute toxicity assays doses of the nanoparticles corresponding to various fractions of the LD50 (1/5, 1/10 and 1/20) were prepared and administered intraperitoneally to each group of mice for 14 consecutive days, whereas the control group was treated with the vehicle (PBS) only. On the 15th day, the animals were sacrificed by cervical dislocation; blood, femur bones and internal organs were collected and used for further evaluation [27]. All animal studies were performed in accordance with the Institutional Animal Ethical Committee’s (IAEC) approval.
2.9. In vivo immunomodulation studies An immunomodulator is a substance that affects the immune system. Lymphocytes were isolated from the blood 14 days after administration of the compound. Its proliferation status was evaluated with the MTT assay as described before, and the status of CD3+, CD4+ and CD8+ cells was determined by flow cytometry and CellQuest Pro [6]. Various biochemical and hematological parameters were also evaluated. Femur bones were collected and cut at the level of epiphyseal plates of the proximal and distal ends of the bone. Bone marrow cells were aspirated after flushing with RPMI media, and cell counts were calculated as described previously [6].
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2.10. Statistical analysis Data were expressed as the mean ± standard deviation (SD) of three replicates and were analyzed using GraphPad PRISM software version 5.0 (GraphPad Software, USA). One-way analysis of variance was used for repeated measurements, and the differences were considered to be statistically significant if P < 0.05. The IC50 values were calculated using the Easy Plot software. 3. Results and discussion 3.1. PST-Gold nanoparticles were prepared and are stable The polysaccharide PST001 isolated from the seed kernels of Ti was found to be pH neutral, and the total sugar content was 98%, as determined by the phenol–sulfuric acid method. PST0001 was purified by gel filtration chromatography, lyophilized and stored. We found that PST001 successfully reduced HAuCl4 to form gold nanoparticles with a deep red color (Fig. 1a). PST001 not only acted as a reducing agent for the production of gold nanoparticles but also served as a capping agent, imparting stability to the PST-Gold nanoparticles and preventing their re-aggregation without the use of any other capping agent. The successful production of PST-Gold nanoparticles was evaluated using UV–vis spectroscopy, which produced the characteristic peak of gold nanoparticles approximately 550 nm (Fig. 1b); PST001 and HAuCl4 failed to produce any characteristic peak from 350 to 750 nm. At an accelerated voltage of 80 kV, TEM evaluation of the nanoparticle size clearly showed that the majority of the particles were circular in shape with an average size of 15–20 nm (Fig. 1c). This result was confirmed using DLS, which also indicated that the particles have an average size of 20 nm (Fig. 1d). Although PST-Gold nanoparticles do not produce any visible aggregation, their stability was evaluated for various time intervals (Fig. 1e) and they remained stable even up to 12 months. Evaluation of nanoparticle stability over a wide range of pH using UV–vis spectroscopy clearly demonstrated a high tolerance for pH change (Fig. 1f) with no significant shift in the position of the peak over a pH range of 1–14. The present study investigated the preparation of gold nanoparticles using the water soluble, anticancer polysaccharide PST001 isolated from the seed kernels of T. indica and further evaluated their biological potential as a cytotoxic agent specific to cancer cells. Water soluble polysaccharides were found to be therapeutically applicable for various pharmacological applications, including the use as drug carriers [28]. Gold nanoparticles have a long history of use in various pharmacological applications [29–31]. The production of PST-Gold nanoparticles using PST001 does not include the use of any hazardous chemicals and environmental toxins and hence is a “green” nanoparticle synthesis process [32]. This process also does not require the use of a capping agent because PST001 itself acts as a capping agent in addition to its role as a reducing agent. PST-Gold nanoparticles have an average size of 20 nm and are stable for up to 1 year with a wide range of pH tolerance. Therefore, they are more stable than commercially available borohydrate- or citrate-reduced gold nanoparticles [15]. 3.2. Anticancer effect of PST-Gold nanoparticles Because PST001 is reported to be an anticancer agent with immunomodulatory properties, the PST-Gold nanoparticles were also evaluated for their anticancer potential on various cancer cell lines. This potential was found to be highly significant (P < 0.001) in all cell lines examined. Cytotoxicity of PST-Gold nanoparticles was also evaluated in the cancer cell lines. Breast cancer cell line MCF7 and leukemia cell line K562 were growth-arrested with
IC50 values of 70.3 ± 1.2 g/ml and 48.9 ± 1.8 g/ml, respectively, after 48 h of incubation with the nanoparticles, whereas the native polysaccharide failed to inhibit 50% of cell growth even at a higher concentration and with a longer incubation period (Fig. 2a and b, and Supplementary Fig. 1a and b) in both cell lines. The cytotoxic potential of the nanoparticles increased in a dose-dependent manner up to 100 g/ml in both cell lines. PST-Gold nanoparticles also exhibited excellent cytotoxic potential against A549 (adenocarcinoma), A375 (melanoma), HepG2 (hepatocellular carcinoma) and HCT116 (colon cancer) cells. IC50 values of the nanoparticles were 53.4 ± 0.9 g/ml and 33.8 ± 1.1 g/ml at 48 h for A549 and A375 cells, respectively, whereas the IC50s for PST001 in the two cell lines were 82.03 ± 1.6 g/ml and 61 ± 1.7 g/ml, respectively, after 72 h of incubation. PST-Gold nanoparticles exhibited a dose-dependent increase in cytotoxicity in A375 cells but reached optimum cytotoxicity at 100 g/ml in A549 cells (Fig. 2c and d, and Supplementary Fig. 1c and d). The cytotoxic potential increased in a dose-dependent fashion up to 10 g/ml for HepG2 cells, and an IC50 value of 6.5 ± 0.5 g/ml was obtained after 48 h of incubation. In HepG2 cells, the IC50 dose of PST001 was 33.7 ± 1.3 g/ml after 72 h of incubation (Fig. 2e, and Supplementary Fig. 1e). In HCT116 cells, PST-Gold nanoparticles also exhibited a dose-dependent increase in cytotoxicity, with an IC50 of 50.07 ± 1.5 g/ml at 48 h, whereas the IC50 of PST001 was 82 ± 1.2 g/ml at 72 h (Fig. 2f, and Supplementary Fig. 1f). Standard citrate-capped gold nanoparticles of 20 nm in size and 1 mM HAuCl4 were found to be nontoxic in all of these cell lines. To evaluate the cytotoxic potential of PST001 in normal cells, an MTT assay was performed on isolated normal lymphocytes. PST-Gold nanoparticles were found not only to be nontoxic to the cells across all concentrations but also strongly immunostimulatory. A proliferative index (PI) of 1.19 was obtained at a concentration of 0.1 g/ml. Standard gold nanoparticles showed little toxicity at higher concentrations but exhibited no immunostimulatory effects (Fig. 2g). Earlier we showed that PST001 alone was much less cytotoxic to cancer cells than PSTGold nanoparticles. Because PST001 was found to be nontoxic to normal cells, the nanoparticles were also checked for cytotoxicity on isolated normal lymphocytes. We found that, in addition to being nontoxic, PST-Gold nanoparticles also exerted immunostimulatory effects with a PI of up to 1.19 on the lymphocytes. Various polysaccharides were previously reported to have both anticancer and immunomodulatory effects [1,3,6]; however, the present study revealed that PST-Gold nanoparticles are more potential than other compounds. 3.3. PST-Gold nanoparticles exert anticancer effects through induction of apoptosis Because PST-Gold nanoparticles exhibited significant cytotoxicity specifically against cancer cells, their mode of cell death induction was evaluated using various apoptotic assays. Membrane blebbing, which is a hallmark of apoptosis, refers to irregular bulges in the plasma membrane of a cell caused by localized decoupling of the cytoskeleton from the plasma membrane. Morphological evaluation of the cell lines treated with PST-Gold nanoparticles at a concentration of 10 g/ml for 48 h using phasecontrast microscopy revealed a decrease in the number of cells exhibiting morphological features of apoptosis, such as distorted shape and membrane blebbing in comparison with the control group (Supplementary Fig. 2). Using acridine orange–ethidium bromide staining, cells treated with PST-Gold nanoparticles showed a change in color from green to yellow/red with associated apoptotic features such as membrane blebbing, nuclear condensation and presence of apoptotic bodies compared to the control (Fig. 3, insert). PST-Gold-induced apoptosis was confirmed by flow cytometry analysis of Annexin V staining. There was a significant
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Fig. 1. Preparation and stability assay of PST-Gold nanoparticles. (a) Photographic image of freshly prepared PST-Gold nanoparticles. (b) UV–vis spectra of PST-Gold nanoparticles, HAuCl4 and PST001. (c) TEM images of PST-Gold nanoparticles, magnified 20,000×. (d) DLS spectra of PST-Gold nanoparticles. (e) Stability assay of PST-Gold nanoparticles in various time intervals and (f) in various pH conditions. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
(P < 0.001) increase in the Annexin V positive cells in all of the cell lines treated with 10 g/ml PST-Gold nanoparticles for 48 h (Supplementary Table 1). In MCF 7 cells, the controls exhibited 2 ± 0.07% Annexin V positive cells, whereas compound treated cells were 41.4 ± 1.5% positive (Fig. 3a and b). A similar pattern was observed for K562, A549 and A375 cell lines, where the control cells were 4.5 ± 0.9%, 11 ± 1.2% and 16 ± 1.1% Annexin V positive and PST-Gold nanoparticle-treated cells were 8.3 ± 0.9%, 35 ± 1.6% and 51 ± 1.9% positive, respectively (Fig. 3c–h). The percentages of apoptotic HepG2 and HCT116 cells significantly increased from 3.5 ± 0.6% and 9.9 ± 1.3% in the controls to 47 ± 2.1% and 32.5 ± 2.2% in nanoparticle-treated cells, respectively (Fig. 3i–l). The enhanced activity exhibited by the nanoparticles compared with the parent polysaccharide might be due to the increased uptake of the particles via endocytosis because of their smaller size and increased surface to volume ratio [33]. 3.4. In vivo evaluation of PST-Gold nanoparticles Even though PST-Gold nanoparticles exhibited significant cytotoxicity against cancer cells and immunomodulatory effects in vitro, their activity in biological systems requires further evaluation. In vivo toxicity of PST-Gold nanoparticles was assessed on 5- to 6-week-old male BALB/c mice. There were no behavioral changes or visible toxicity symptoms upon ip administration of PST-Gold up to a concentration of 2000 mg/kg; hence, the LD50 was taken to be 2000 mg/kg. After the administration of the compound at different concentrations for 14 consecutive days, animals were sacrificed by cervical dislocation and various parameters were analyzed. None of the PST-Gold nanoparticle-treated animals from any
of the groups exhibited weight loss throughout the duration of the experiment. Various biochemical and hematological parameters of the sacrificed animals were assessed for any toxicity induced by the nanoparticles (Supplementary Table 2). There was an increase in the RBC and WBC count in the groups administered with the compound, regardless of the concentration, which again reinforced the immunostimulatory effects of PST-Gold nanoparticles. There was no significant variation in any of the common biochemical parameters for the compound-treated mice compared with the control; moreover, no toxicity was observed to be associated with the administration of the compound. Hematoxylin–eosin staining was performed on the livers, kidneys, lungs, hearts and spleens of the sacrificed animals to evaluate any pathological changes associated with compound administration at the organ level. No significant pathological changes were observed with the heart, lung and spleen in any of the PST-Gold-treated mice compared with the control group (Supplementary Fig. 3). PST-Gold nanoparticles administered at a concentration of 100 mg/kg appeared to have no effect on the liver and kidney (Fig. 4c and d), and mice treated with 200 and 400 mg/kg compound exhibited slight cytological abnormalities. The hepatocytes appeared to be slightly swollen in morphology with enlarged cytoplasm and normal nuclei in the mice given 200 mg/kg compound (Fig. 4f); the shape of the cells appeared to be irregular at the higher concentration (Fig. 4h), but the nuclei remained normal. Although the glomeruli appeared normal for the mice given 200 mg/kg PST-Gold nanoparticles, the cells lining the renal tubules showed slight changes with a swollen morphology (Fig. 4e). Similar to the changes in the glomeruli, the tubular cells also appeared larger with a smaller lumen (Fig. 4g).
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Fig. 2. MTT assay on cancer cell lines and lymphocytes. (a) Cytotoxicity profile on 48-h incubation of PST-Gold nanoparticles, PST001 and standard citrate capped gold nanoparticles on MCF-7 cells, (b) on K562 cells, (c) on A549 cells, (d) on A375 cells, (e) on HepG2 cells, (f) on HCT116 cells and (g) on isolated normal lymphocytes at 72 h of incubation. Results are expressed as the mean ± SD.
Compound-treated mice exhibited a significant (P < 0.001) increase in the lymphocyte proliferation status both at the time of sacrifice (0 h) and at 72 h after in vitro incubation in comparison with the control group (Fig. 5a). Although all concentrations of the nanoparticles showed significant immunomodulatory activity, the mice given with 100 mg/kg PST-Gold nanoparticles showed maximum lymphocyte proliferation with PIs of 1.8 ± 0.01 and 1.6 ± 0.08 at 0 h and 72 h, respectively. Immunophenotyping of lymphocyte subsets with CD markers (CD3, CD4 and CD8) showed an increase in the proportion of the T lymphocyte population in the PSTGold nanoparticle-treated mice compared with the control group
(Fig. 5b). Mice given 100 mg/kg compound exhibited a significant increase in all of the T lymphocyte subsets, with PIs of 48.7 ± 4.5%, 35.7 ± 4% and 11.2 ± 2% for CD3, CD4 and CD8 subtypes, respectively. In comparison, the PIs in the control group were 22.8 ± 3.1%, 24.5 ± 3% and 3.3 ± 0.3% for CD3, CD4 and CD8 subtypes, respectively. The elevated T cell population serves as an index for the immunostimulatory property of PST-Gold nanoparticles. The bone marrow cellularity was determined by counting the number of bone marrow cells from the femur bones of the sacrificed animals. The group administered with 100 mg/kg PST-Gold showed the maximum bone marrow cell count at 8 × 106 cells/femur, whereas
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Fig. 3. Apoptotic evaluation of cell lines treated with 10 g/ml PST-Gold nanoparticles for 48 h by Annexin V–FITC staining assay. The insert figure shows acridine orange–ethidium bromide staining images of the same on corresponding cell lines. MCF 7 cells (a) control and (b) PST-Gold. K562 cells (c) control and (d) PST-Gold. A549 cells (e) control and (f) PST-Gold. A375 cells (g) control and (h) PST-Gold. HepG2 cells (i) control and (j) PST-Gold. HCT116 cells (k) control and (l) PST-Gold. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
the control group produced 4.8 × 106 cells/femur. Notably, the bone marrow cell counts were significantly higher (P < 0.001) in the PST-Gold treated mice than in the control group across all concentrations (Fig. 5c). Acute toxicity assay performed on nanoparticle-treated BALB/c mice did not show any lethal effect, and the LD50 was taken
to be 2000 mg/kg. Histopathological evaluation of various organs after hematoxylin–eosin staining demonstrated no pathological symptoms for the heart, lung and spleen, but slight changes were observed with the liver and kidney in mice administered with 200 and 400 mg/kg nanoparticles. However, analysis of various biochemical parameters revealed no abnormality compared with
Fig. 4. Light microscopic images of H&E staining of various organs of BALB/c mice after 14 days administration of PST-Gold nanoparticles and normal saline. Control (a) kidney and (b) liver. 100 mg/kg PST-Gold (c) kidney and (d) liver. 200 mg/kg PST-Gold (e) kidney and (f) liver. 400 mg/kg PST-Gold (g) kidney and (h) liver.
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Fig. 5. Immunomodulatory effects of PST-Gold nanoparticles on BALB/c mice after 14 days of administration of the compound. (a) Lymphocyte proliferation status of the sacrificed animal at the hour of sacrifice (0 h) and after 72 h. (b) Immunophenotyping for the status of lymphocyte subsets. (c) Bone marrow cellularity from the bone marrow cells of femur bones. Results are expressed as the mean ± SD. Statistically significant differences are at **P < 0.01, ***P < 0.001, ns is the non-significant, as compared with the control group.
the control mice. The slight pathological abnormalities observed with the liver and kidney at higher concentrations of nanoparticles were expected to be reversible. The in vivo immunomodulatory effects of PST-Gold nanoparticles were further demonstrated by significant increases in the lymphocyte proliferation status, bone marrow cell count and T lymphocyte population in mice treated with the nanoparticle for 14 days relative to the controls. Conventional chemotherapeutic agents often suppress the immune system of the host [34], which warrants the search for anticancer agents that are nontoxic to normal cells. Gold nanoparticles have been evaluated in diverse applications, including in vitro assays, in vitro and in vivo imaging, cancer therapy and drug delivery. Tumor-targeting technologies that exploit gold’s inherent biocompatibility are being developed to deliver drugs directly into cancerous tumors. Additionally, simple, cost-effective and sensitive diagnostic tests are being developed for the early detection of prostate and other cancers. Furthermore, application of gold nanoparticles in radiotherapy has been reported recently. Roa et al. reported that gold nanoparticle sensitizes prostate cancer cells to radiation by regulating the cell cycle [35]. Gold nanoparticle could also provide advantages in terms of radiation dose enhancement [36], as reported by Wan et al. Therefore, PST-Gold nanoparticle needs to be evaluated for its various applications in cancer treatment and management. 4. Conclusions The preparation of PST-Gold nanoparticles using the anticancer polysaccharide PST001 was found to be of immense therapeutic value because the particles exhibited both immunomodulatory and cytotoxic properties. PST-Gold nanoparticles exerted its cytotoxicity in tumor cells through the induction of apoptosis and were found to be nontoxic to normal tissues in BALB/c mice. Although slight
pathological abnormalities were observed based on H&E staining at higher concentrations of the nanoparticle, the groups given lower doses of the nanoparticle were found to be unaffected. Immunostimulatory effects of PST-Gold nanoparticles on BALB/c mice were more pronounced 14 days after administration of the compound, as demonstrated by a significant increase in bone marrow cell count and CD3/4/8 counts. The data presented here suggest that PST-Gold nanoparticle may be used as an effective anticancer agent with strong immunomodulatory potential. Acknowledgements We greatly acknowledge the Council of Scientific and Industrial Research (CSIR), Govt. of India, for the research fellowship; Kerala State Council for Science, Technology and Environment (KSCSTE), Govt. of Kerala, for the financial support; and the National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, for the DLS analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2012.11.046. References [1] V.E. Ooi, F. Liu, Curr. Med. Chem. 7 (2000) 715. [2] V.R. Sinha, R. Kumaria, Pharm. Int. J. 224 (2001) 19. [3] M.J. Manu, S.R. Aravind, V. Sheeja, S. Mini, T.T. Sreelekha, Mol. Med. Rep. 5 (2012) 489. [4] F. Liu, V. Ooi, S.T. Chang, Life Sci. 60 (1997) 763. [5] G. Franz, Planta Med. 55 (1989) 493. [6] S.R. Aravind, M.J. Manu, V. Sheeja, B. Prabha, T.T. Sreelekha, Sci. World J. (2012) 361382.
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